1. Field of the Invention
The present invention relates to a method for two-dimensional separation of the constituents of a mixture, using a capillary electrophoresis technique, carried out in a capillary or an equivalent system such as a microchip-type system. This method, especially adapted to the separation of mixtures of electrically charged polymers (synthetic or natural), such as mixtures of proteins or of peptides, is for example useful for analyzing the constituents of such mixtures.
2. Description of the Related Art
“Two-dimensional” separation techniques consist in carrying out the separation of the constituents of a mixture according to two distinct successive criteria. Thus, these methods generally implement a first step which consists in separating the constituents of the mixture under first separation conditions, and then a second step consisting in subjecting all or some of the separated fractions obtained at the end of the first step to a further separation under new conditions. The two successive separation steps used in these methods generally implement two separations of different types, based on two distinct characteristics of the constituents to be separated. These two successive separation steps are referred to as “orthogonal” with respect to one another.
The term “two-dimensional separation” comes from the fact that, originally, this type of separation denotes a separation carried out on supports actually having two dimensions, such as plates. Thus, for example, two-dimensional chromatography consists in loading a sample of mixture onto a plate, in carrying out an elution of the mixture by means of a first eluent in a first direction of the plate, and then in eluting each of the separated fractions of the mixture (aligned according to the first direction) using a second eluent in a direction which is orthogonal to the first direction, whereby a separation of the constituents in the two dimensions of the plate is obtained. Similarly, two-dimensional electrophoresis techniques on a gel layer have been developed, comprising a first electrophoretic separation of the compounds of a mixture by migration according to a first direction of the gel, followed by a second migration in an orthogonal direction, under other electrophoresis conditions. These techniques are widely used, especially in the field of the separation and analysis of mixtures of proteins. In this case, two-dimensional electrophoresis techniques on gel, called (IEF/SDS-PAGE), have in particular been developed, which are suitable for the separation of proteins or peptides, and in which the first separation is carried out by isoelectric focusing (IEF), which allows separation according to the isoelectric point in a first direction of the gel, and the second step consists of electrophoretic migration in an orthogonal direction, carried out in the presence of a polyacrylamide gel and sodium dodecyl sulfate (SDS-PAGE). For further information concerning two-dimensional electrophoresis techniques, and in particular techniques of the (IEF/SDS-PAGE) type, reference may in particular be made to the article by P. A. Haynes, S. P. Gygi, D. Figeys, R. Aebersold, in Electrophoresis, 19, 1862-1871 (1998).
By extension, the term “two-dimensional” separation has subsequently been extended to any method using two successive steps of one-dimensional separation, in which the second step consists in separating the compounds of at least one of the fractions separated during the first step. By analogy with the techniques on a two-dimensional support, the second separation step is referred as being carried out “according to an orthogonal direction”, even when no two-dimensional support is concretely used.
Various two-dimensional separation techniques are currently known, which couple two (or more) successive “one-dimensional” separation steps, such as chromatography or electrophoresis steps. By way of examples of two-dimensional separation techniques of this type, mention may in particular be made of the techniques described in the work High Performance Capillary Electrophoresis, Chemical Analysis Series, vol. 146, No. 17, 581-612 (1998) or those described by J. M. Hille, A. L. Freed, and H. Watzig, in Electrophoresis, 22, 4035-4052 (2001).
Especially, two-dimensional separation techniques using at least one step of capillary electrophoresis in their separation steps have been developed. In this context, certain authors have in particular described the coupling of two successive capillary electrophoresis steps. In this respect, reference may e.g. be made to the articles by Mark R. Shure in Anal. Chem. 71, 1645-1657 (1999), or by Deepa Mohan and Cheng S. Lee in Electrophoresis 23, 3160-3167 (2002).
These two-dimensional separation methods appear, a priori, to be attractive, especially since they seem to be capable of combining the advantages of two-dimensional separation with the particularly marked effectiveness of electrophoretic separation. However, in practice, these methods generally require the use of complex and most commonly expensive devices, and the separation carried out is not always up to the quality expected.
Especially, techniques using two successive steps of capillary electrophoresis currently consist in coupling two capillaries, so as to be able to carry out the two electrophoreses in distinct media. This coupling of two capillaries is generally difficult to carry out, and it requires coupling devices which are often expensive.
Furthermore, as underlined in particular by Shure et al., in Anal. Chem 71, 1645-1657 (1999), the use of these coupling devices generally affects the quality of the separation produced, resulting in particular in phenomena of peak broadening which can be limited only with great difficulty. Furthermore, the coupling of two capillaries most commonly involves sampling steps, during which part of the compounds to be analyzed can be lost, or involves dilution processes which prove in particular to be prejudicial in terms of the sensitivity of detection of the compounds separated, most particularly when the initial mixture to be analyzed is a dilute medium.
Thus, the techniques of two-dimensional separation by coupling of two successive capillary electrophoresis steps that are currently known prove to be unsatisfactory in terms of effectiveness and/or of cost. Given these drawbacks, this type of two-dimensional separation is very rarely used in practice.